Master of Science / Department of Diagnostic Medicine and Pathology / Megan Niederwerder / Evidence has shown that the gastrointestinal microbiome plays an important role in response to infectious disease. Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial microbiome work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70 days post-infection (dpi). However, it remained uncertain if microbiome characteristics could predispose response to viral challenge. The purpose of this study was to determine if microbiome characteristics present at the time of viral challenge were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces on 0 dpi from pigs identified as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced PRRSV replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of the high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. Our results indicate that both microbiome diversity and composition prior to virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection. We followed this study by investigating the microbiome characteristics that are present after co-infection and the role of the microbiome in subclinical infections. Microbiome analysis at 3 and 6 weeks post-infection showed no significant difference between high and low growth rate pigs. The results from both exploring the impact that the initial microbiome has on outcome as well as examining the trends in the microbiome during the post-infection period demonstrate that microbiome pre-infection composition may play a larger role in the outcome of subclinical disease in pigs than microbiome composition during viremia or after viral clearance.

Porcine Reproductive and Respiratory Syndrome (PRRS) is, globally, one of the costliest of diseases to the pig industry. Despite enormous efforts, methods such as vaccination strategies and herd management have failed to fully control the disease. Exploiting the genetic variation in host response could be included as part of a multifaceted approach to mitigate the devastating impact of this disease. Establishing the presence of genetic variation and its underlying genetic architecture are key to implementing genomic selection, which is considered a viable and safe long-term disease control strategy. This thesis explores the effect of natural PRRSV outbreaks on the reproductive performance of sows, and the underlying genetic influences on it. Litter records were available from two farms, where Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) outbreaks had been confirmed using ELISA. One farm had full pedigree information, but for both farms 60K SNP genotypes were available. In both farms, performance records could be partitioned into an epidemic and non-epidemic phase using a previously established threshold method. The partitioning also identified a period of high reproductive failure not coinciding with a diagnosed PRRSV outbreak on one farm. This period was isolated and analysed separately. Linear mixed models were used to explore both genetic and non-genetic factors contributing to differences in reproductive performance associated with the two phases. This analysis identified five disease indicator traits identified showing significant differences (>95% CI) in least squares means between the epidemic and non-epidemic phase. These were the number of mummified, stillborn, dead and alive piglets per litter and the fraction of the total born dead. Alternative statistical models that accounted for differences in the severity of the individual PRRSV outbreaks were also considered throughout. Despite differences in the estimates associated with different models and farms, in general very low heritability estimates were obtained for these disease indicator traits during the non-epidemic phase, whereas the traits were found moderately heritable during the epidemic phase. Two genome wide association analyses methods were used to explore the distribution of the genetic effects throughout the genome: Family-based Score Test for Association (FASTA) and Genome-wide Rapid Analysis using Mixed Model and Regression (GRAMMAR). In addition, regional associations were studied using Regional Heritability Mapping (RHM). Associations were then further characterised using Measured Genotype (MG) analyses. Genome-wide significant associations were identified for five SNPs and one region. The regional association spans the region previously identified in an experimental challenge experiment of growing pigs, in association with viral load and weight gain. Different patterns of linkage disequilibrium (LD) are observed which may explain why this study and others failed to find single SNP effects at this location. One genome wide significant SNP on SSC15 was found between two previously identified SNPs associated with PRRSV mortality. Five further putative SNP associations are indicated by RHM and subsequent measured genotype analysis, two of which flank previously reported associations and indicate an epistatic effect, observed in several traits. In summary, this study showed that reproductive performance of sow is considerably reduced during PRRSV outbreaks and the genetics of the sow significantly affects variance in survival and mortality. Several novel genomic regions associated with the reproductive performance of sows in the absence and during PRRSV outbreaks have been identified in this study. In addition to these, the results suggest the region on SSC4 previously associated with PRRSV viral load and weight gain may also affect foetal mortality. These results demonstrate the potential for genomic selection to be used to mitigate PRRSV related reproductive losses, the greatest financial exposure faced by the pig industry. In addition, RHM is directly shown to capture genetic variance, where single SNP methods fail to identify an effect, highlighting the usefulness of this tool as a method to identify genomic regions with significant effect on production traits.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases facing the global swine industry. Viraemia profiles of PRRS virus challenged pigs reflect the severity and progression of infection within the host and provide crucial information for subsequent control measures. In this thesis we analyse the largest longitudinal PRRS viraemia dataset from an in-vivo experiment, and corresponding immune measures in the form of cytokine data and neutralising antibodies. In the PRRS Host Genetic Consortium (PHGC) trials, pigs were challenged with one of two PRRSV isolates (NVSL and KS06, respectively). In Chapter 2 we derive a statistical description of the temporal changes in viraemia and determine the influence of diverse factors (e.g. PRRSV strain, pig genetic background, resistance genotype, etc.) on viraemia profiles. The well-established methodology of linear mixed modelling with a repeated measures model and fitting a linearized Wood’s function, a gamma-type function, is applied to the viraemia dataset. The virus isolate had a significant impact on the viraemia profiles which was captured by statistically significant differences in model parameters via both statistical methods. The more virulent NVSL isolate had higher early viraemia predictions and a faster rate of decline than KS06. In line with previous studies the WUR “resistance” genotype, associated with lower AUC viraemia found in previous studies, also resulted in lower viraemia predictions in the statistical models. The typical time trends of the viraemia profiles were a rise to a peak followed by a period of decline with dynamics and magnitude influenced by the virus isolate. Both uni and bimodal viraemia profiles were observed. The Wood’s model appeared a suitable candidate model for the data associated with uni-modal profiles and captured the time trends concisely in only three model parameters which also had a biological relevance. Overall the best fitting Wood’s model (y=atbe-ct) was when there was a random effect in Wood’s parameters b and c. Bimodal profiles significantly reduced the model fit, particularly in the later phase of infection resulting in large model residuals. However bimodal profiles did not impact upon the significance of the differences between the LSM repeated measures estimates nor the LSM linearized Wood’s model parameter estimates. The longitudinal viraemia measures from the PRRSV challenge experiment revealed substantial differences in the viraemia profiles between hosts infected with the same PRRSV challenge dose, pointing to considerable variation in the host response to PRRSV infections. In Chapter 3 we provide a suitable mathematical description of all viraemia profiles with biologically meaningful parameters for quantitative analysis of profile characteristics. The Wood’s function and a biphasic extended Wood’s function were fit to the individual profiles using Bayesian inference with a likelihood framework in Chapter 3. Using maximum likelihood inference and numerous fit criteria, we established that the broad spectrum of viraemia trends could be adequately represented by either uni-or biphasic Wood’s functions. Three viraemic categories emerged: cleared (uni-modal and below detection within 42 days post infection(dpi)), persistent (transient experimental persistence over 42 dpi) and rebound (biphasic within 42 dpi). The convenient biological interpretation of the model parameters estimates, allowed us not only to quantify inter-host variation, but also to establish common viraemia curve characteristics and their predictability. The convenient biological interpretation of the model parameters estimates, allowed us not only to quantify inter-host variation, but also to establish common viraemia curve characteristics and their predictability, which were utilized in subsequent quantitative genetic analyses to identify genomic regions associated with these new resistance traits. The Bayesian approach for curve fitting in Chapter 3 led to better model fits than the classical linear mixed models approach of Chapter 2. Furthermore in Chapter 4 we explored the association between the observed PRRS viraemia profile characteristics and the corresponding measures of the immune response in the form of: neutralising antibody (nAb) cross protection data and longitudinal cytokine profiles. Statistical analysis of the profile characteristics revealed that persistent profiles were distinguishable already within the first 21 dpi, whereas it is not possible to predict the onset of viraemia rebound. Analysis of the neutralizing antibody (nAb) data indicated that there was a ubiquitous strong response to the homologous PRRSV challenge, but high variability in the range of cross-protection of the nAbs. Persistent pigs were found to have a significantly higher nAb cross-protectivity than pigs that either cleared viraemia or experienced rebound within 42 dpi. We determined the typical features and time trends of each cytokine profile, examined the associations between cytokines, and characterised the cytokine response. A stronger association was found in the direction of cytokines driving the ensuing viraemia characteristics as opposed to vice versa. It was found that viraemia class differences were best captured in the anti-viral cytokine IFNA and also the chemokine CCL2, furthermore these key cytokines were the most strongly associated with viraemia measures. The breadth of the cytokine responsiveness was associated with viral profile class and genetic background but not the WUR genotype. The statistical categorization of pigs from each PHGC trial through model fitting provides a critical basis for the generation of new desirable host phenotypes, and of potential use in the genetic selection of pigs with favourable infection traits. Our study provides novel insights into the nature and degree of variation of hosts’ responses to infection as well as new informative traits for subsequent genetic and modelling studies.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-10-05T14:03:09Z No. of bitstreams: 1 texto completo.pdf: 298725 bytes, checksum: 7bee1975846b19c9ac200de64bd75213 (MD5) / Made available in DSpace on 2016-10-05T14:03:09Z (GMT). No. of bitstreams: 1 texto completo.pdf: 298725 bytes, checksum: 7bee1975846b19c9ac200de64bd75213 (MD5) Previous issue date: 2003-04-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O circovírus suíno (PCV) é um agente amplamente distribuído na Europa, América do Norte e sul da Ásia. O PCV é um pequeno vírus de cadeia simples de DNA (17 nm) que foi reconhecido, a partir da década de 90, como um patógeno de suíno. Dois tipos de PCV tem sido caracterizados e designados PCV tipo 1 (PCV1) e PCV tipo 2 (PCV2). O PCV1 foi primeiramente isolado em 1974 como um contaminante persistente da linhagem de células PK-15 de rim de suíno (ATCC CCL 31) e a cepa de PCV isolada de células PK-15 tem sido bem caracterizada. O PCV1 é considerado como um vírus não patogênico, enquanto que a infecção de um suíno pelo PCV2 é normalmente associada ao desenvolvimento de Síndrome Multissistêmica Pós-Desmame (PMWS), em animais de 5 a 12 semanas de idade, e ao tremor congênito (CT), que acomete animais no período neonatal. A PMWS é uma nova doença emergente de suínos, caracterizada clinicamente por dispnéia progressiva, aumento dos nódulos linfáticos e patologicamente caracterizada por uma ampla extensão de lesões inflamatórias. Recentemente, pesquisadores da EMBRAPA iniciaram um estudo da PMWS em leitões, mas no Brasil a presença do PCV ainda não é reconhecida oficialmente. O objetivo desse trabalho foi (1) padronizar técnicas de diagnóstico para o genoma e antígeno do PCV, assim como anticorpos contra o agente; (2) avaliar a susceptibilidade de diferentes linhagens celulares ao PCV; (3) diagnosticar a infecção do PCV em suínos da Zona da Mata de Minas Gerais; (4) isolar o PCV de amostras positivas. O PCV, proveniente de tecidos de animais normais e com diagnóstico de CT, foi isolado em células SK6 e analisadas por PCR. O padrão de bandas foi o mesmo encontrado em células PK15 contaminadas com PCV2, gentilmente cedidas pela EMBRAPA. Os oligos usados diferenciavam o PCV1 do PCV2. Todos os leitões de maternidade testados por PCR foram positivos para o PCV2. Porém, em 59 animais de abate testados por PCR não foi observada a presença do PCV. No teste de susceptibilidade as células PK15, SK6, VERO e MDCK foram susceptíveis ao PCV, mas somente as PK-15 estavam persistentemente infectadas. No ensaio de imunofluorescência indireta, foi utilizado um conjugado anti-IgG suína previamente padronizado e anticorpos contra PCV foram identificados em soros de 24 em 44 animais de abate testados e nenhum anticorpo foi encontrado nos animais com diagnóstico de CT positivos para PCV2 por PCR. Com esses resultados podemos concluir que os 24 suínos de abate soropositivos entraram em contato com o agente e desenvolveram a infecção em alguma fase durante o estagio de produção. A ausência de soropositivos entre os leitões recém nascidos, aliada a presença de infecção, pode ser explicada pela incapacidade de produção de anticorpos por esses animais neste estágio de desenvolvimento. Estudos adicionais da epidemiologia e da imunologia de infecções pelo PCV são necessários para o melhor entendimento e efetivo controle das doenças associadas a esse vírus. / Porcine circovirus (PCV) is thoroughly an agent distributed in Europe, North America and south of Asia. PCV is a small virus of simple chain of DNA (17 nm) that was recognized, starting from the decade of 90, as a swine pathogen. Two types of PCV have been characterized and designated PCV type 1 (PCV1) and PCV type 2 (PCV2). PCV1 was isolated firstly in 1974 as a persistent contaminant of the lineage of cells PK-15 of swine kidney (ATCC CCL 31) and the stump of isolated PCV of cells PK-15 has been well characterized. PCV1 is considered as a non- pathogenic virus, while the infection of a swine for PCV2 is usually associated to Post weaning Multisistemic Wasting Syndrome (PMWS), in animals from 5 to 12 weeks of age, and to the congenital tremor (CT), that attack animals in the neonatal period. PMWS is a new emergent disease of swine, clinically characterized by progressive dispnea, increase of the lymphatic nodules and pathologically characterized by a wide extension of inflammatory lesions. Recently, researchers of EMBRAPA began a study of PMWS in pigs, but in Brazil the presence of PCV is not still recognized officially. The objective of that work was (1) to standardize diagnosis techniques for the genome and antigen of PCV, as well as antibodies against the agent; (2) to evaluate the susceptibility of different cellular lineages to PCV; (3) to diagnose the infection of PCV in swine of the Zona da Mata of Minas Gerais; (4) to isolate PCV of positive samples. PCV, originating from tissues of normal animals and with diagnosis of CT, it was isolated in SK6 cells and analyzed by PCR. The pattern of bands was the same found in contaminated cells PK15 with PCV2, kindly by EMBRAPA. The used oligos differentiated PCV1 of PCV2. All the pigs of maternity tested by PCR were positive for PCV2. However, in 59 slaughtering animals tested by PCR, PCV was not found. In susceptibility test, PK15, SK6, VERO and MDCK cells were susceptible for both PCV but only PK15 cells were persistently infected. Anti-PCV antibodies were found to be positive in 54,5% of slaughtering animals serum and any anti-PCV antibody was found in animals with clinical CT. Rapid and accurate diagnosis and removal of disease animals from farms, combined with good husbandry practices, would appear to be the only current method of controlling losses attributable to PCV2 infections. However, additional studies into the epidemiology and immunology of PCV infections are now required if better understanding and eventual control of the disease syndromes associated with these viruses are to be achieved. / Dissertação importada do Alexandria.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine. To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV. We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5. / Ph. D.

L'apport des anticorps immunomodulateurs ciblant PD-1, PD-L1 et CTLA-4 ont récemment révolutionné la prise en charge thérapeutique du cancer. Cependant, seule une minorité de patients développent des réponses objectives à ces traitements. Par conséquent, de nouvelles innovation thérapeutiques sont nécessaires afin d'augmenter l'immunogénicité des tumeurs et de surmonter la résistance à la thérapie contre les anticorps immunomodulateurs. Les propriétés oncolytiques de certains virus peuvent être exploitées afin de permettre un amorçage de l'immunité anti-tumorale. Différents virus oncolytiques (OVS) sont actuellement en développement clinique intense en combinaison avec des thérapies par anticorps immunomodulateurs. Nous avons trouvé qu'un vaccin viral pédiatrique disponible dans le commerce a des propriétés oncolytiques. Ce virus pédiatrique peut tuer directement les cellules cancéreuses, avec des caractéristiques de mort cellulaire immunogène. De plus, ce virus a des propriétés pro-inflammatoires et peut activer la voie NF-kB indépendamment des voies de danger cellulaire (TLR et IRF). Ces propriétés biologiques in vitro se traduisent in vivo en une activité anti-tumorale. L'Injection intra-tumorale du vaccin a des effets anti-tumoraux directs mais également à médiation immunitaire. De façon intéressante, dans des modèles de souris immunocompétentes porteuses de tumeurs murines, l'injection intra-tumorale de vaccin a un effet synergique avec des anti CTLA-4 permettant la guérison de 100% des souris. Les vaccins sont des produits pédiatriques et adultes de grade clinique. Par conséquent, des stratégies de vaccination in situ par injection intra-tumorale pourraient être rapidement mises en clinique / Immune checkpoint targeted therapies against PD-1, PD-L1 and CTLA-4 are currently revolutionizing cancer care. However, only a minority of patients develop objective responses with these treatments. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors in order to overcome resistance to immune checkpoint blockade therapy. Oncolytic properties of common viruses can be exploited for the priming of anti-tumor immunity and such oncolytic viruses (OVs) are currently in intense clinical development in combination with immune checkpoint targeted therapies. We have found that commercially available virus vaccines do have oncolytic properties. These pediatric vaccine virus can directly kill cancer cells with features of immunogenic cell death. Moreover, it has pro-inflammatory properties and can activate the NF-Kb pathway in a toll-like receptor and IRF3 independent manner. These in vitro biological properties translate in vivo into anti-tumor activity. Intra-tumoral vaccine therapy has anti-tumor effects which are partly immune mediated. Interestingly, in immunocompetent murine pediatric tumor models, intra-tumoral injection overcome resistance and synergize with immune checkpoint targeted therapy. Vaccines are pediatric and adult clinical grade products. Therefore, in situ intra-tumoral immunization strategies could be implemented quickly in the clinic

Background: In this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the sameherd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs. Results: At the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N. Conclusions: Only one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated.

An investigation of the distribution of porcine reproduction and respiratory syndrome virus (PRRSV) and factors associated with the presence of PRRSV in Ontario from 2004 – 2007 was conducted. Surveys on the presence of clinical signs associated with PRRS, management practices, animal suppliers, and herd location were administered to the managers of 458 PRRSV positive herds and 61 PRRSV negative herds. Open reading frame (ORF) 5 of the PRRSV genome was sequenced from herds with PRRSV. PRRSV positive herds were compared to PRRSV negative herds. Management practices associated with being PRRSV positive were: not washing animal- and feed-delivery vehicles, feed-delivery and animal-transport vehicles visiting multiple herds at one time, allowing a truck driver to enter the barn, not requiring visitors to shower prior to farm entry, and not utilizing all-in all-out flow in gilt and finisher barns. Specific PRRSV restriction fragment length polymorphism (RFLP) genotypes of the ORF5 gene were compared with clinical signs. Herds with RFLP type ‘1-undetermined-4’, ‘1-undetermined-2’ and 1-3-4 were associated with clinical signs in sows and 2-6-2 was associated with finisher mortality compared to herds with vaccine virus. Additionally, genotypes 1-3-4 and 1-8-4 increased in frequency during this study. The between-herd PRRSV similarity of genome and clinical signs were compared. Abortions and stillbirths were associated with similarity in genetic sequences between herds. This relationship did not extend to those herds where vaccine virus was identified. Patterns in space and time of herds with different RFLP types of PRRSV were investigated after accounting for ownership. There was weak evidence to suggest local spread the genotype 1-3-4. The association between genetic similarity and proximity in space, time, ownership, animal, and semen suppliers was tested. Significant correlation was detected for distances up to 30 km. After controlling for ownership, only small associations between breeding stock and semen suppliers and genetic similarity of PRRSV were found. The spread of PRRSV among herds in Ontario cannot be attributed to any one factor. However, similarity in ownership between herds was a key variable indicating that movement of animals, personnel, and vehicles among herds must be measured in future investigations of PRRSV dynamics. / Ontario Pork and the Canada-Ontario Research and Development (CORD) Program and the OMAFRA/University of Guelph agreement

A host can adopt two strategies when facing infection: resistance, where host immune responses prevent or reduce pathogen replication; or tolerance, which refers to all mechanisms that reduce the impact of the infection on host health or performance. Both strategies may be under host genetic control, and could thus be targeted for genetic improvement. Although there is ample evidence of genetic variation in resistance to infection, there is limited evidence to suggest that individuals also differ genetically in tolerance. Furthermore, although resistance and tolerance are typically considered as alternative host defense mechanisms, relatively little is known about the genetic relationship between them and how they change together over time and jointly determine infection outcome. In this thesis, two datasets from experimental challenge infection experiments were considered for investigating tolerance genetics: Porcine Reproductive & Respiratory Syndrome (PRRS), an endemic viral disease which causes loss of growth and mortality in growing pigs; and Listeria monoctyogenes (Lm), a bacterium which causes food-borne infections in mammals. The two datasets differed substantially in size and genetic structure; the PRRS dataset consists of thousands of records from outbred commercial pig populations, whereas the Listeria dataset comprises much fewer records from genetically diverse highly inbred strains of a mice as a model species. The aims of this thesis were to: 1) Identify if genetic variation in host tolerance to infection exists, with case studies in PRRS and listeria, using conventional reaction-norm methodology; 2) Identify if host tolerance, along with resistance, changes longitudinally as infection progresses; 3) Identify whether the WUR genotype is associated with tolerance slope; 4) Analyse the dynamic relationship between host performance and pathogen load over the time-course of infection by examining the relationship at different stages of infection using GWAS; 5) Develop novel trajectory methodology to offer insight into health-infection dynamics, and identify whether there is genetic variation in trajectories; 6) Develop novel trajectory-derived phenotypes that analyse changes in host performance with respect to changes in pathogen load, as an alternative to tolerance, and identify whether genetic variation exists. This study found that conventional reaction-norm methodology is limited to capture genetic variation in tolerance in outbred populations without measures of performance in the absence of infection. However, by utilising repeated longitudinal data on the same dataset, stages of infection (early, mid and late) were defined for each individual, based on host pathogen load. Using these stages of infection, genetic variation in tolerance was identified over all stages of infection and at mid to late stage of infection. Genetic correlation between resistance and tolerance was strong and positive over all stages of infection, and evidence suggested that resistance and tolerance may be under pleiotropic control. Furthermore, this research found that genetic correlations between resistance and growth changed considerably over time, and that individuals who expressed high genetic resistance early in infection tended to grow slower during that time-period, but were more likely to clear the virus by late stage, and thus recover in growth. However, at mid-late stage of infection, those with high virus load also had high growth, indicating potential epidemiological problems with genetic selection of host resilience to infection. Furthermore, genome wide association studies for pathogen load and growth associated with different stages of infection did not identify novel genetic loci associated with these traits than those previously reported for the whole infection period. By adopting conventional methodology, this study found genetic variation in tolerance of genetically diverse mouse strains to Lm and pigs to PRRS, despite statistical problems. The relationship between resistance and tolerance indicated that both traits should be considered in genetic selection programs. By adopting novel trajectory analysis, this study demonstrated that level of expression of resistance and tolerance changed throughout the experimental infection period and, furthermore, that expression of resistance, followed by tolerance, determined survival of infection. Survivors and non-survivors followed different infection trajectories, which were partially determined by genetics. By deriving novel phenotypes from trajectories that explained changes in growth in relation to change in pathogen load at specific time points, and applying these to the PRRS data, this study did not identify genetic variation in these phenotypes. The genetic signal in these phenotypes may have been masked by the fact that individuals were likely at different stages of infection. In summary, this study has shown that genetic improvement of tolerance, in addition to resistance may be desirable, but could be difficult to achieve in practice due to shortcomings in obtaining accurate and unbiased tolerance estimates based on conventional reaction-norms. Infection trajectories have proven to be a promising tool for achieving an optimally timed balance between resistance and tolerance, but further work is needed to incorporate them in genetic improvement programs.

Transduction of melanoma cells with the aim to induce avidine expression on tumor cell surface was studied. Subsequently the method enabling quantification of binding of ligands to the cell surface was developed.